Evolution and mechanism of spectral tuning of Blue-absorbing visual pigments in butterflies

The eyes of flower-visiting butterflies are often spectrally highly complex with multiple opsin genes generated by gene duplication, providing an interesting system for a comparative study of color vision. The Small White butterfly, Pieris rapae, has duplicated blue opsins, PrB and PrV, which are expressed in the blue (λmax = 453 nm) and violet receptors (λmax = 425 nm), respectively. To reveal accurate absorption profiles and the molecular basis of the spectral tuning of these visual pigments, we successfully modified our honeybee opsin expression system based on HEK293s cells, and expressed PrB and PrV, the first lepidopteran opsins ever expressed in cultured cells. We reconstituted the expressed visual pigments in vitro, and analysed them spectroscopically. Both reconstituted visual pigments had two photointerconvertible states, rhodopsin and metarhodopsin, with absorption peak wavelengths 450 nm and 485 nm for PrB and 420 nm and 482 nm for PrV. We furthermore introduced site-directed mutations to the opsins and found that two amino acid substitutions, at positions 116 and 177, were crucial for the spectral tuning. This tuning mechanism appears to be specific for invertebrates and is partially shared by other pierid and lycaenid butterfly species

Human blue cone opsin regeneration involves secondary retinal binding with analog specificity

Human color vision is mediated by the red, green, and blue cone visual pigments. Cone opsins are G-protein-coupled receptors consisting of an opsin apoprotein covalently linked to the 11-cis-retinal chromophore. All visual pigments share a common evolutionary origin, and red and green cone opsins exhibit a higher homology, whereas blue cone opsin shows more resemblance to the dim light receptor rhodopsin. Here we show that chromophore regeneration in photoactivated blue cone opsin exhibits intermediate transient conformations and a secondary retinoid binding event with slower binding kinetics. We also detected a fine-tuning of the conformational change in the photoactivated blue cone opsin binding site that alters the retinal isomer binding specificity. Furthermore, the molecular models of active and inactive blue cone opsins show specific molecular interactions in the retinal binding site that are not present in other opsins. These findings highlight the differential conformational versatility of human cone opsin pigments in the chromophore regeneration process, particularly compared to rhodopsin, and point to relevant functional, unexpected roles other than spectral tuning for the cone visual pigmentsPeer ReviewedPostprint (author's final draft

Functional Properties of Visual Pigments using A1 and A2 Chromophore : From Molecules to Ecology

The first event in vision is the absorption of a photon by a visual pigment molecule in a retinal photoreceptor cell. Activation of the molecule triggers a chemical amplification cascade, which finally leads to a change in the membrane potential of the cell. However, a visual pigment molecule may also be spontaneously activated by thermal energy. The resulting electrical response is identical to that caused by a photon. Such false light signals form a background noise limiting the detection of dim light. The absorption spectrum of a visual pigment (its ability to use different wavelengths of light) and its propensity for thermal activation both depend on the minimum amount of energy required for activation (the activation energy Ea). These properties of the pigment can be tuned on an evolutionary time scale by changes in the amino acid sequence of the protein part (the opsin) or on a physiological time scale by changing the light-sensitive cofactor bound to the opsin, the chromophore. The latter option is accessible only to poikilothermic vertebrates having two alternative chromophores (retinal A1 and A2). In this thesis, functional consequences of the A1-A2 exchange were investigated. In the first part, the relation between the changes of the absorption spectrum and the activation energy was quantitatively measured in several species of amphibians and fishes using both chromophores. The A2-induced shift of the absorption spectrum towards longer wavelengths was found always to correlate with a decrease in Ea. Later investigations have confirmed that decreasing Ea increases the rate of thermal activations. Thus the switch from A1 to A2 in the same opsin gives a more red-sensitive but noisier pigment. Against this background, the second part of the thesis investigates chromophore usage in eight populations of nine-spined sticklebacks (Pungitius pungitius) from different light environments. The amino acid sequence of the rods was found to be identical in all populations, implying that variations in spectral sensitivity depended only on the A1:A2 ratios. The cone absorption spectra also suggested that the variation within each cone class was due to varying chromophore proportions alone. The differences between populations could not be consistently explained as adaptations to the different light environments. However, an important and quite unexpected result was that the same individual could have quite different chromophore proportions in rods and cones (more A2 in cones). This shows that there are mechanisms by which chromophore proportions in different photoreceptors can be regulated much more selectively than previously thought. Since pigment noise is sensitivity-limiting mainly in dim light, it may be suggested that cones (working mainly in brighter light) can better afford using the noisy A2 chromophore to shift their spectral sensitivities for a better match to a long-wavelength photic environment.Näkötapahtuma alkaa, kun verkkokalvon fotoreseptorisoluissa sijaitseva näköpigmenttimolekyyli absorboi fotonin. Molekyylin aktivoituminen käynnistää kemiallisen vahvistusketjun, jonka lopputuloksena solun kalvojännite muuttuu. Näköpigmenttimolekyyli voi kuitenkin aktivoitua myös spontaanisti lämpöenergian vaikutuksesta (termisesti), synnyttäen sähköisen vasteen joka on täysin samanlainen kuin fotonin aiheuttama. Tällaiset väärät valosignaalit muodostavat taustakohinan, joka rajoittaa heikkojen valojen havaitsemista. Näköpigmentin absorptiospektri (sen kyky käyttää valon eri aallonpituuksia) ja sen taipumus aktivoitua termisesti riippuvat molemmat aktivaation vaatimasta minimienergiamäärästä (ns. aktivaatioenergiasta Ea). Pigmentin ominaisuuksia voidaan säätää joko evolutiivisella aikaskaalalla proteiiniosan (opsiinin) aminohapposekvenssiä muuttamalla tai fysiologisella aikaskaalalla opsiiniin sidotun valoherkän kofaktorin, ns. kromoforin, vaihdolla. Jälkimmäinen optio on vain vaihtolämpöisillä selkärankaisilla, joilla on käytössään kaksi vaihtoehtoista kromoforia (retinaali A1 ja A2). Tässä väitöskirjassa tutkittiin A1-A2-vaihdon funktionaalisia seurauksia. Ensimmäisessä osassa mitattiin kvantitatiivisesti absorptiospektrin ja aktivaatioenergian muutosten suhdetta useilla sammakko- ja kalalajeilla. Todettiin että A2:een liittyvä absorptiospektrin siirtyminen pitempiin aallonpituuksiin korreloi aina Ea:n laskun kanssa. Myöhemmät tutkimukset ovat vahvistaneet, että Ea:n alentaminen lisää termisten aktivaatioiden määrää. A1-kromoforin vaihtaminen A2:een samassa opsiinissa antaa siis punaherkemmän mutta kohinaisemman pigmentin. Tätä taustaa vasten väitöskirjan toisessa osassa tutkittiin kromoforin käyttöä kahdeksassa, eri valoympäristöissä elävässä kymmenpiikkipopulaatiossa (Pungitius pungitius). Sauvasolujen opsiinien aminohapposekvenssi todettiin identtiseksi kaikissa populaatioissa, joten spektraaliherkkyyden vaihtelu johtui yksinomaan vaihtelevista A1:A2 suhteista. Myös tappisolujen absorptiospektrit viittasivat siihen, että kunkin tappiluokan sisäinen vaihtelu johtui vain kromoforisuhteista. Populaatioiden välisiä eroja ei pystytty johdonmukaisesti selittämään adaptaatioina eri valoympäristöihin. Sen sijaan tärkeä ja täysin odottamaton tulos oli, että saman yksilön sauvoissa ja tapeissa saattoi olla aivan eri kromoforisuhteet (tapeissa enemmän A2). Tämä osoittaa, että on mekanismeja joilla eri reseptoreiden kromoforisuhteita voidaan säätää paljon yksilöidymmin kuin on tiedetty. Koska pigmenttikohina rajoittaa näön herkkyyttä lähinnä heikossa valossa, voidaan ajatella, että nimenomaan tappien spektraaliherkkyyksiä on varaa siirtää A2:lla paremmin vastaamaan keltaisen järven valospektriä, ilman että kohinasta johtuva hinta on liian korkea

Functional characterization of spectral tuning mechanisms in the great bowerbird short-wavelength sensitive visual pigment (SWS1), and the origins of UV/violet vision in passerines and parrots

BackgroundOne of the most striking features of avian vision is the variation in spectral sensitivity of the short wavelength sensitive (SWS1) opsins, which can be divided into two sub-types: violet- and UV- sensitive (VS & UVS). In birds, UVS has been found in both passerines and parrots, groups that were recently shown to be sister orders. While all parrots are thought to be UVS, recent evidence suggests some passerine lineages may also be VS. The great bowerbird (Chlamydera nuchalis) is a passerine notable for its courtship behaviours in which males build and decorate elaborate bower structures.ResultsThe great bowerbird SWS1 sequence possesses an unusual residue combination at known spectral tuning sites that has not been previously investigated in mutagenesis experiments. In this study, the SWS1 opsin of C. nuchalis was expressed along with a series of spectral tuning mutants and ancestral passerine SWS1 pigments, allowing us to investigate spectral tuning mechanisms and explore the evolution of UV/violet sensitivity in early passerines and parrots. The expressed C. nuchalis SWS1 opsin was found to be a VS pigment, with a λmax of 403 nm. Bowerbird SWS1 mutants C86F, S90C, and C86S/S90C all shifted λmax into the UV, whereas C86S had no effect. Experimentally recreated ancestral passerine and parrot/passerine SWS1 pigments were both found to be VS, indicating that UV sensitivity evolved independently in passerines and parrots from a VS ancestor.ConclusionsOur mutagenesis studies indicate that spectral tuning in C. nuchalis is mediated by mechanisms similar to those of other birds. Interestingly, our ancestral sequence reconstructions of SWS1 in landbird evolution suggest multiple transitions from VS to UVS, but no instances of the reverse. Our results not only provide a more precise prediction of where these spectral sensitivity shifts occurred, but also confirm the hypothesis that birds are an unusual exception among vertebrates where some descendants re-evolved UVS from a violet type ancestor. The re-evolution of UVS from a VS type pigment has not previously been predicted elsewhere in the vertebrate phylogeny

Avian visual pigments: Characteristics, spectral tuning, and evolution

Birds are highly visual animals with complex visual systems. In this article, we discuss the spectral characteristics and genetic mechanisms of the spectral tuning of avian visual pigments. The avian retina contains a single type of rod, four spectrally distinct types of single cone, and a single type of double cone photoreceptor. Only the single cones are thought to be involved in color discrimination; double cones are thought to be involved in achromatic visual tasks, such as movement detection and pattern recognition. Visual pigment opsin protein genes in birds are orthologous to those in other vertebrates and have a common origin early in vertebrate evolution. Mechanisms of spectral tuning in the different classes of avian cone visual pigments show similarities in most instances to those in other vertebrates. The exception is the ultraviolet/violet (SWS1) class of pigments; phylogenetic evidence indicates that the ancestral vertebrate SWSI pigment was ultraviolet sensitive (UVS), with different molecular mechanisms accounting for the generation of violet-sensitive (VS) pigments in different vertebrate classes. In birds, however, UVS visual pigments have re-evolved from an ancestral avian VS pigment by using a novel molecular mechanism not seen in other vertebrate classes. This has occurred independently in four of the 14 avian orders examined to date, although the adaptive significance of this is currently unknown

Far-Red Absorbing Rhodopsins, Insights From Heterodimeric Rhodopsin-Cyclases

The recently discovered Rhodopsin-cyclases from Chytridiomycota fungi show completely unexpected properties for microbial rhodopsins. These photoreceptors function exclusively as heterodimers, with the two subunits that have very different retinal chromophores. Among them is the bimodal photoswitchable Neorhodopsin (NeoR), which exhibits a near-infrared absorbing, highly fluorescent state. These are features that have never been described for any retinal photoreceptor. Here these properties are discussed in the context of color-tuning approaches of retinal chromophores, which have been extensively studied since the discovery of the first microbial rhodopsin, bacteriorhodopsin, in 1971 (Oesterhelt et al., Nature New Biology, 1971, 233 (39), 149–152). Further a brief review about the concept of heterodimerization is given, which is widely present in class III cyclases but is unknown for rhodopsins. NIR-sensitive retinal chromophores have greatly expanded our understanding of the spectral range of natural retinal photoreceptors and provide a novel perspective for the development of optogenetic tools.Peer Reviewe

Directed Evolution of Gloeobacter violaceus Rhodopsin Spectral Properties

Proton-pumping rhodopsins (PPRs) are photoactive retinal-binding proteins that transport ions across biological membranes in response to light. These proteins are interesting for light-harvesting applications in bioenergy production, in optogenetics applications in neuroscience, and as fluorescent sensors of membrane potential. Little is known, however, about how the protein sequence determines the considerable variation in spectral properties of PPRs from different biological niches or how to engineer these properties in a given PPR. Here we report a comprehensive study of amino acid substitutions in the retinal binding pocket of Gloeobacter violacaeus rhodopsin (GR) that tune its spectral properties. Directed evolution generated 70 GR variants with absorption maxima shifted by up to +/- 80 nm, extending the protein’s light absorption significantly beyond the range of known natural PPRs. While proton pumping activity was disrupted in many of the spectrally shifted variants, we identified single tuning mutations that incurrred blue and red shifts of 42 nm and 22 nm, respectively, that did not disrupt proton pumping. Blue-shifting mutations were distributed evenly along the retinal molecule while red-shifting mutations were clustered near the residue K257, which forms a covalent bond with retinal through a Schiff base linkage. Thirty-four of the identified tuning mutations are not found in known microbial rhodopsins. We discovered a subset of red-shifted GRs that exhibit high levels of fluorescence relative to the wild-type protein

The Microbial Opsin Family of Optogenetic Tools

The capture and utilization of light is an exquisitely evolved process. The single-component microbial opsins, although more limited than multicomponent cascades in processing, display unparalleled compactness and speed. Recent advances in understanding microbial opsins have been driven by molecular engineering for optogenetics and by comparative genomics. Here we provide a Primer on these light-activated ion channels and pumps, describe a group of opsins bridging prior categories, and explore the convergence of molecular engineering and genomic discovery for the utilization and understanding of these remarkable molecular machines.National Institutes of Health (U.S.) (TR01)Bill & Melinda Gates FoundationSimons FoundationDamon Runyon Cancer Research FoundationMcKnight FoundationRobert MetcalfeHelen S. Boylan Foundatio